Because synaptic transmission and plasticity is the molecular basis that underlies drug abuse and addiction (as well as learning and memory), it is essential to gain a detailed understanding of the molecular mechanism of synaptic plasticity and the pharmacological actions of drugs on synaptic strength and activities. There is a lack of information about loading and chemical makeup of single synaptic vesicles because no reliable methods exist to probe the chemical compositions of such a small biochemical container (approximately 50-nm in diameter containing approximately 10-19 L of solutions). To address this challenge, we propose to integrate a set of complementary technologies we have developed - single vesicle manipulation, single-molecule detection, and nanofluidics - for probing the chemical compositions of individual synaptic vesicles. Our specific short-term goals for this application is thus the following: (1) Direct imaging and optical manipulation of single 50-nm vesicles in a suitably designed and fabricated nanofluidic system, (2) Develop appropriate reaction schemes to dye tagged the contents of single 50-nm vesicles, followed by separation and single-molecule detection of the separated species, and (3) Study variations of the composition, if they exist, among individual synaptic vesicles and ascertain whether there may be subpopulations of vesicles. The main goal of this R21 proposal, therefore, is to integrate the technologies we have developed and demonstrate their ability to analyze the chemical makeup of individual 50-nm synaptic vesicles. With this demonstration, we have laid down the technological foundation for achieving the long-term goal of answering many of the hypotheses on synaptic transmission and plasticity, including: (1) Alteration of synaptic strength by differential presynaptic loading of transmitters into vesicles, (2) The co-localization of multiple fast transmitters within the same vesicle and chemical variations among individual synaptic vesicles, (3) The quantitation of the absolute number of neurotransmitters contained within a single synaptic vesicle, and (4) The direct experimental testing of the Dale hypothesis by analyzing the chemical contents of single synaptic vesicles from different synaptic sites of the same neuron.